The zebrafish has become the model system of choice for a growing number of investigators interested in understanding mechanisms of vertebrate development, disease and evolution. The three principal investigators on this multiple-Principal Investigator proposal have each established the Targeting Local Lesions IN Genomes (TILLING) methodology for identifying N-ethyl-N-nitrosourea (ENU)-induced mutations in specific genes of interest in zebrafish using Cel1 detection, and have used this approach to identify nonsense or splice site mutations predictive of strong or complete loss of function of 43 genes. The approach involves screening for unique mutations in a large library of randomly ENU-mutagenized, cryopreserved fish that has been built independently in each of our labs. We wish to make these valuable resources available to the zebrafish community. In this 3-year grant, we propose to identify loss-of-function mutations (defined for our purposes as mutations that create premature stop codons or that disrupt splice sites) in 120 genes of interest to the members of the zebrafish community and to make them available via submission to the Zebrafish International Resource Center (ZIRC). Establishing a TILLING consortium between the three groups reduces redundancy of effort and increases the chances of identifying deleterious mutations in target genes. Whereas separately, our libraries are predicted to contain loss-of-function mutations in between 20% and 58% of the genes we screen, combined, they are expected to contain loss-of-function mutations in over 80% of targets. In Aim 1 we propose to screen target genes sequentially at the three locations until one or more loss-of-function mutations are identified. Potential TILLING targets will be submitted by members of the community and will be ranked by a 10-member external advisory board according to criteria such as high biomedical relevance and inaccessibility to other reverse genetics methods. In Aim 2 we propose to recover these mutants, to do a preliminary phenotypic characterization and to provide them first to the requester and then, within six months of recovery, to the wider zebrafish community via ZIRC. Finally, in Aim 3 we propose to explore massively parallel sequencing of PCR-amplified targets from our ENU-mutagenized libraries as a higher-throughput alternative to our current TILLING methodology. PUBLIC HEALTH RELEVANCE: Vertebrate model organisms such as the zebrafish, in which gene function can be understood through the detailed analysis of mutant phenotypes, provide important insights into mechanisms of human development and disease. In the past grant period we adapted TILLING, a methodology to find mutations in genes of interest, to the zebrafish, and built resources that can allow us to find loss-of-function mutations in a large fraction of the genes in the zebrafish genome. We now propose to use TILLING to identify mutations in 120 genes that are of importance to biomedical research, and to make these mutants available to the zebrafish community as rapidly as possible.